Departamento de Bioquímica e Imunologia, FMRP - Universidade de São Paulo-USP, Ribeirão Preto, São Paulo CEP 14049-900, Brazil.
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom.
Int J Biol Macromol. 2018 Dec;120(Pt A):279-287. doi: 10.1016/j.ijbiomac.2018.07.116. Epub 2018 Jul 21.
Microbial β-galactosidases (EC 3.1.2.23) have applications in the production of galacto-oligosaccharides, which are established prebiotic food ingredients. The β-galactosidase from Bacillus subtilis (YesZ) was expressed as a heterologous protein in Escherichia coli, and presented an optimum activity at pH 6.5 and 40 °C. The catalytic constants K and V of the enzyme were 8.26 mM and 1.42 μmol·min·mg against pNP-β-d-galactopyranoside, respectively. Structural characterization revealed that YesZ is a homotrimer in solution, and homology modeling suggested that the YesZ conserves a Cys cluster zinc binding site. Flame photometry experiments confirmed the presence of bound zinc in the recombinant enzyme, and YesZ activity was inhibited by 1 mM zinc, copper and silver ions. Transgalactosylation activity of YesZ was observed with the synthetic substrate p-NP-βGal in the presence of a d-xylose acceptor, producing a β-d-galactopyranosyl-(1 → 4)-d-xylopyranose disaccharide. Analysis of this disaccharide by MALDI-ToF-MS/MS suggested a β-1,4 glycosidic linkage between a non-reducing galactose residue and the xylose. The β-galactosidase YesZ from B. subtilis is a candidate for enzymatic synthesis showing favorable thermostability (with residual activity of 50% after incubation at 30 °C for 25 h) and transgalactosylation activity.
微生物β-半乳糖苷酶（EC 3.1.2.23）在半乳糖低聚糖的生产中具有应用，这些低聚糖是已确立的益生元食品成分。枯草芽孢杆菌的β-半乳糖苷酶（YesZ）作为一种异源蛋白在大肠杆菌中表达，在 pH 6.5 和 40°C 时表现出最佳活性。该酶对 pNP-β-d-半乳糖吡喃糖苷的催化常数 K 和 V 分别为 8.26 mM 和 1.42 μmol·min·mg。结构特征表明 YesZ 在溶液中是三聚体，同源建模表明 YesZ 保守了一个 Cys 簇锌结合位点。火焰光度法实验证实重组酶中存在结合锌，YesZ 活性被 1mM 锌、铜和银离子抑制。YesZ 在合成底物 p-NP-βGal 存在下具有转半乳糖基化活性，用 d-木糖作为受体，生成 β-d-半乳糖吡喃糖基-(1 → 4)-d-木吡喃糖苷二糖。通过 MALDI-ToF-MS/MS 对该二糖进行分析表明，非还原半乳糖残基和木糖之间存在β-1,4 糖苷键。枯草芽孢杆菌的β-半乳糖苷酶 YesZ 是一种具有有利热稳定性（在 30°C 孵育 25 小时后残留活性为 50%）和转半乳糖基化活性的酶合成候选物。
